Samples were collected at Wind Cave National Park on multiple different trips. As the team goes deeper and deeper into the cave, the goal is to actively trace the types of microorganisms (fungi, bacteria, and archaea) throughout the cave. The process to obtain genus- and species-level identification of these samples utilizes PCR amplification of different sections of their genomic DNA followed by sequencing analysis. The objective of this project is to make a genetic map of the Wind Cave microbial system that includes public tour routes as well as deep wild cave regions, including previously reported lake extremophiles. This effort will determine how genetically unique or isolated the lake system is and what influence surface microbes and anthropogenic contamination from cave explorers may have had on the subterranean microbiome. An initial phylogenetic tree showing the relatedness of the identified microorganisms is presented. This project is based upon work supported by the Iowa Space Grant Consortium under NASA Award No. 80NSSC20M0107.